The goals of this project are to investigate the role of altered ion homeostasis and ion signaling in cell injury and protective adaptation. Cells have an inherent ability to protect themselves from injury caused by environmental stress and other types of injury, and tumor cells may upregulate these pathways to enhance their survival. One series of studies are investigating the mechanisms involved in protective adaptation (preconditioning). Brief intermittent periods of stress (ischemia, H2O2, osmotic stress, LPS etc.), termed preconditioning (PC), provide protection against injury during a subsequent longer period of stress). Current studies to elucidate the signaling pathways that are responsible for PC have shown the following. 1. Protein kinase C (PKC) is involved in PC. Activators of PKC mimic preconditioning and inhibitors of PKC block PC. Furthermore, hearts from mice that overexpress PKC-epsilon exhibit less injury, as if they were constitively preconditioned. 2. Phosphoinositide-3-kinase (PI3-kinase) is upstream of PKC. PC causes phosphorylation of PKB, the kinase directly downstream of PI3-kinase and PC induced phosphorylation of PKB is blocked by wortmannin, an inhibitor of PI3-kinase. Wortmannin also blocks the protective effects of preconditioning, but does not block the protection induced by DOG, a direct activator of PKC. DOG does not cause phosphorylation of PKB. Furthermore, wortmannin blocks the PC induced translocation of PKC-epsilon. 3. PI3-kinase activation leads to activation of nitric oxide synthase. PC leads to an increase in nitric oxide production, as measured by an increase in total nitrate + nitrite, and this increase is blocked by wortmannin Immunohistochemistry shows that preconditioning leads to an increase in phospho-eNOS in the myocytes and this increase is blocked by wortmannin. 4. PC leads to phosphorylation of GSK-3?, an effect that is blocked by wortmannin, suggesting involvement of PI3-kinase. Since phosphorylation inactivates GSK-3?, we tested whether inhibition of GSK-3? with Li+ would mimic preconditioning. Perfusing hearts with Li+ significantly reduced injury, suggesting that PC induced inhibition of GSK-3? is an important mediator of protection. 5. The cardioprotective actions of diazoxide, a selective opener of the mitochondrial K+ ATP channel, are mediated by generation of a pro-oxidant environment. Because reactive oxygen species (ROS) are recognized as important intracellular signaling molecules, and have been implicated in ischemic preconditioning, we examined diazoxide-induced ROS production in adult cardiomyocytes. Cells treated with 50 mM diazoxide showed a 173% increase in ROS production, relative to baseline, respectively. 5-hydroxydecanoate was found to attenuate the diazoxide-induced increase in ROS generation. The diazoxide-induced increase in ROS was abrogated also by the addition of either the antioxidant N-acetylcysteine (NAC) or N-mercaptopropionylglycine. We also examined the ability of NAC to block the protective effects of diazoxide using the perfused rat heart. After 20 minutes of global ischemia and 20 minutes of reflow, hearts perfused with 100 ?M diazoxide prior to ischemia showed significantly improved post ischemic contractile function relative to untreated hearts (84% versus 29% of initial LVDP, respectively). Hearts treated with diazoxide in the presence of 4 mM NAC recover 53% of initial LVDP, whereas hearts treated with NAC alone hearts recovered 46% of preischemic function. A second series of studies are investigating the basis of gender differences in the response to cell injury.. In hearts from three different transgenic mouse models (Na-Ca overexprssor, ?2-adrenergic overexpressor, phospholamban KO), which all would be expect to have an increase in sarcoplasmic reticulum calcium, we find that male but not females have poorer recovery of function following stress. The functional recovery of hearts from bilateral ovariectomized female transgenic mice was significantly worse than that of non-ovariectomized female transgenics and similar to that of male transgenic mice, suggesting that estrogen counters the effects of overexpression of the Na/Ca exchanger. Although we do not typically observe male/female differences in response to injury in wild type control mice, such differences can be observed if we raise the extracellular calcium or treat hearts with isoproterenol, conditions which would elevate cell calcium. We directly measured sarcoplasmic reticulum calcium in male and female hearts treated with isoproterenol using a 19F NMR sensitive high Kd calcium indicator, and preliminary data suggest that isoproterenol results in a smaller increase n SR Ca2+ in females than in males. Since protection in females is mediated via estrogen and estrogen activates both endothelial and inducible nitric oxide synthase (eNOS and iNOS), we pretreated male and female ?2-overexpressor hearts with either 1 ?mol/L of the nonspecific NOS inhibitor, L-NAME, or 100 nmol/L of the specific iNOS inhibitor, 1400W. There was no effect of L-NAME on male ?2-overexpressors or of 1400W on male ?2-overexpressors. Ischemic injury in female b2AR overexpressors was exacerbated by treatment with L-NAME, but not 1400W, implying that it was eNOS, not iNOS, which was protective. We also found increased generation of nitrates and nitrites in hearts from female mice. To investigate the effect of NO on SR Ca2+ we treated perfused hearts with the NO donor, SNAP, and measured SR Ca2+ with the 19F NMR indicator. We found that SNAP significantly reduced SR Ca2+ from 1 mM to 0.7 mM. By western blot we found that SNAP resulted in nitrosylation of tyrosine in SERCA. Taken together these data suggest that altered NO signaling leading to modification of SERCA and SR Ca2+ are important mediators of gender differences in response to injury.